Lignin is a complex heterogeneous aromatic polymer which waterproofs and reinforces the walls of certain plant cells.
Lignin is formed by the polymerization of free radicals derived from monolignols such as paracoumarylic, coniferylic and sinapylic alcohols (Higuchi, 1985, in Biosynthesis and degradation of wood components (T. Higuchi, ed), Academic Press, Orlando, Fla. pp. 141-160).
Lignins have a large variation in their relative monolignol levels, as a function of the species and the different tissues within the same plant.
This variation is probably due to and controlled by the different activities and specificities of substrates, of enzymes necessary for the biosynthesis of lignin monomers (Higuchi, 1985, mentioned above).
Beyond its role in the structure and development of plants, lignin represents a major component of the terrestrial biomass and assumes a great economic and ecological significance (Brown, 1985, J. Appl. Biochem. 7, 371-387; Whetten and Sederoff, 1991, Forest Ecology and Management, 43, 301-316).
With regard to the exploitation of the biomass, first and foremost it should be noted that lignin is a factor limiting the digestibility and nutritional yield of fodder crops. In fact, it is clearly demonstrated that the digestibility of fodder crops by ruminants, is inversely proportional to the lignin level of these plants, the nature of the lignins also being a determining factor in this phenomenon (Buxton and Roussel, 1988, Crop. Sci., 28, 553-558; Jung and Vogel, 1986, J. Anin. Sci., 62, 1703-1712).
Among the principle fodder crops in which it would be useful to reduce the level of lignins, there can be mentioned: alfalfa, fescue, maize, fodder used for silage . . . .
It should also be noted that high lignin levels are in part responsible for the limited quality of sunflower cakes intended for cattle food and the reduction in the viability of certain seeds in the horticultural domain.
It can also be emphasized that the intense lignification which occurs during the storage of plant organs after harvest, rapidly renders crops such as asparagus, yams, carrots, etc. unfit for consumption.
Moreover, it should also be noted that more than 50 million tons of lignins are extracted from ligneous material each year in the production of paper pulp in the paper industry. This extraction operation which is necessary to obtain cellulose is costly in terms of energy and secondarily polluting due to the chemical compounds employed in the extraction and which find their way into the environment (Dean & Eriksson, 1992, Holzforschung, 46, 135-147; Whetten and Sederoff, 1991, mentioned above).
To reduce the proportions of lignins (which depending on the species represent 20 to 30% of the dry material) to a few percent (2 to 5%) would represent an increase in yield, a substantial saving (chemical products) and would contribute to the improvement of the environment (reduction in pollution). Given the scale on which ligneous material is used, these effects would have extremely significant repercussions. In this case, the species concerned could be the poplar, the eucalyptus, Acacia mangium, the Casuarina genus and all the angiosperms and gymnosperms used in the production of paper pulp.
In the two fields considered, it is clear that the reduction of lignin levels must be moderate in order for the plant (or tree) to retain its characteristics of rigidity and its normal architecture, as the lignins which reinforce the cell walls play an important role in maintaining the upright habit of plants.
Natural variations in the levels of lignins observed in nature for the same species (difference which can range up to 6-8% of the dry mass between individuals) permit the reductions mentioned above.
The resistance to degradation of lignin, as well as the difficulties encountered in its extraction, are probably due to the complex structure of this polymer constituted by ether and carbon--carbon bonds between the monomers, as well as to the numerous chemical bonds existing between lignin and other components of the cell wall (Sarkanen and Ludwig, 1971, in Lignins: Occurrence, Formation, Structure and Reactions (K. V. Sarkanen and C. H. Ludwig eds) New York: Wiley--Interscience, pp. 1-18).
Starting from cinnamoyls-CoA, the biosynthesis of lignins in plants, is carried out in the following manner: ##STR1##
One approach, using the genetic engineering route, to try to reduce the level of lignins in plants, would consist of inhibiting the synthesis of one of the enzymes of the biosynthesis chain of these lignins indicated above.
A technique which is particularly appropriate within the scope of such an approach, is that of using anti sense mRNA capable of hybridizing with the mRNA coding for these enzymes, and as a consequence, of inhibiting, at least partially, the production of these enzymes from their corresponding mRNA.
Such an anti sense strategy, carried out using the gene coding for CAD in tobacco, was the subject of the European Patent Application No. 584 117, describing the use of anti sense mRNA capable of inhibiting the production of lignins in the plants by hybridizing with the mRNA coding for CAD in these plants.
The results at the level of the plants transformed in this way demonstrate a reduction in the activity of CAD, but paradoxically, the lignin levels show no change. Additional studies indicate that the lignins of the transformed plants are different from the control lignins, because the cinnamylic aldehydes are directly incorporated in the lignin polymer.